Axons utilize cues found along migration pathways for navigation to their final destinations. Early steps in zebrafish motor axon migration require drwanka gene activity. In diwanka embryos, primary RoP motor axons migrate too far within the spinal cord and fail to exit, while CaP-like secondary axons exit the cord ectopically. Studies showing that diwanka activity is required cell non-autonomously for motor axon migration suggest that diwanka may be a local guidance cue. However, the identity of this gene and the molecular pathways in which it act are unknown. The objective of this proposal is to gain an understanding of the molecular mechanisms that underlie vertebrate motor axon migration through an analysis of the diwanka mutation. To accomplish this goal, the following specific aims have been developed: (1) To test whether the ability of motor axons to recognize spinal cord exit points is dependent upon diwanka activity, the migration of GFP-expressing motor axons will be examined in vivo through time lapse analysis. Comparison of migration behaviors in wild-type and mutant embryos will reveal whether loss of activity affects motor axon recognition of spinal cord exit points. These experiments will determine if diwanka is also required for this step of motor neuron migration. (2) To test the hypothesis that secondary motor axon migration requires diwanka activity in adaxial cells, the ability of labeled wild-type adaxial cells to rescue secondary motor axon migration in chimeric mutant embryos will be examined. If this hypothesis is correct, it will demonstrate that primary and secondary motor axons rely upon guidance cues provided by the same cell type for their migration. However, if this hypothesis is incorrect, these experiments will reveal the identity of the cell type that provides diwanka activity required for secondary motor axon migration. (3) To identify the molecular function of diwanka, this gene will be cloned. This will reveal the pathway in which diwanka acts, allow the identification of interacting factors and lead to the discovery of homologs that promote motor axon guidance in other vertebrate species.